Location: Houston, Texas2011 Annual Report
1a. Objectives (from AD-416)
Objective 1: Determine gene expression in human lactating mammary epithelium. Subobjective 1A: Determine the pattern of mammary epithelial gene expression using milk fat globule mRNA from delivery through the first 4 weeks of lactation. Compare these results with those in mothers of premature infants and teenage mothers over a similar period of time. Subobjective 1B: Characterize the mRNA response to exogenous lactogenic hormones. Objective 2: Characterize inbred mouse strains for lactation performance, gene expression and weight gain among offspring in lean and obese animals, making use of a cross-fostering design where appropriate. Subobjective 2A. Identify genes in which strain-dependent differences in mammary gland gene expression, and SNP haplotype, are correlated with strain-dependent differences in milk production, lactation persistence, mammary gland development, or milk composition. Subobjective 2B. Determine the extent to which genes identified from the whole genome scan and microarray work described in 2A are responsible for the lactation defect in mice with maternal obesity. Objective 3: Study the effect of nutrients on mammary gland development and function in mouse models. Define the critical window for effects on mammary gland development and function. Subobjective 3A1. Determine effect of exposure to low protein diet by analyzing mammary gland development, milk production, and milk composition, as well as gene expression and gene promoter methylation in mammary gland tissue of dams exposed to diets with low protein content during gestation. Subobjective 3A2. Use a mouse model for tissue-specific alteration of Dnmt1 levels to confirm role of DNA methylation in effects of low protein diet on mammary gland development. Subobjective 3B. Define critical window for effect of low protein diet on mammary gland function by limiting nutritional intervention to specific developmental windows. Subobjective 3C. Determine impact of low protein diet on genetic variants for mammary gland development and lactation capacity as identified in objective 2.
1b. Approach (from AD-416)
Children's Nutrition Research Center researchers will determine gene expression in human lactating mammary epithelium by isolating mRNA from human colostrum or milk over the first 4 weeks post partum and the expression arrays measured to determine the relative gene express over this period of time. Data from groups of mothers will be assessed to prove or disprove our hypotheses. A variety of potential lactogenic hormones will be administered short term (over 3 days) to normal women with established lactation between 6 and 12 weeks post partum. The hormones initially to be tested are prolactin, cortisol, and IGF-1. Breast milk will be collected every 3 hr and RNA isolated for measurement of expression of mRNA expression using microchip technology. The data will be compared to that already obtained from similar studies in women prior to and following the administration of recombinant human growth hormone. Additionally, a panel of lactation traits will be measured in 32 inbred strains of mice. The data from these measurements will be used as phenotype data in combination with whole genome SNP data to conduct a statistical association analysis across the entire mouse genome. The Viable yellow agouti (Avy) mouse will be used as a model of maternal obesity. Gene expression will be determined by microarray analysis of mammary tissue samples collected from obese and lean Avy females during early lactation. Genes that are differentially expressed between lean and obese females will then be compared to the list of genes identified to test for overlap. The lactation traits, as well as gene expression and epigenetic profiles will be measured in transgenic animals containing the conditional allele for Dnmt1 (dnmt1-lox2) and a mammary gland specific Cre recombinase to determine the effects of deletion in the mammary gland of Dnmt1. The data will be compared to those of low protein diets.
3. Progress Report
In Obj. 1, we completed the collection/analysis of milk samples obtained from normal mothers from delivery through 6 weeks post partum and have carried out the initial determination of the messenger RNA in the milk (mRNA carries the messages as to which proteins should be made, expression array analyses) but have only begun to do the more complex job of interpreting massive amounts of data. We focused on galactose (a component of milk sugar) and lactose (milk sugar) and observed that the genes involved in the synthesis of a compound UDP-galactose and its transport are low at birth and increase dramatically by 96 hours after birth. This suggests that these pathways may be rate limiting for milk sugar (lactose) production, a major determinant of milk volume. The collection from mothers with premature infants, teenage mothers, and obese mothers has been difficult. We have determined that our Obj. 1B will not be possible to execute. Despite a publication demonstrating the use of human prolactin, we have found no company willing to provide rh-prolactin for human study since it has no profitable commercial use. Without prolactin, the comparisons with glucocorticosteroid and insulin become of lesser importance. We have moved to the measurement of micro RNAs in human milk. Micro-RNAs are small pieces of RNA containing 18 to 30 nucleotides (or building blocks of RNA) and are thought to play a unique regulatory role in the turning on/off of genes and thus the production of a specific protein. In Obj. 2 we analyzed milk and mammary tissue samples collected from 32 inbred strains during the first 10 days of lactation. Data from this analysis is being used to map the locations of genes that could cause variations in milk production. We analyzed and summarized data collected on the weight gain of litters nursed by mouse dams in each of the 32 strains. In Obj. 3 we determined that the mouse model system intended to result in mammary-gland-specific inactivation of genes of interest is not functioning as intended. Alternative research methods had to be identified and used. We used an alternative cell-culture-based method to inactivate genes of interest. This method resulted in inactivation of the gene of interest. From preliminary analysis we concluded that there is no major adverse effect of inactivation of the gene of interest, but we observed subtle changes in cell behavior that, if substantiated, could have effects on the development and function of the mammary gland. Further analyses are ongoing. Additionally, we have determined that the defective animal-model, although not functioning as intended, can still give insight into the role of the gene of interest in mammary function, and we continue to analyze the collected samples. To prepare for the analysis of DNA methylation in the gene inactivation model and animals fed experimental diets (low protein, LP) during pregnancy, we set-up the experimental procedures to be used in these experiments. The ADODR monitors project activities by visits, review of purchases of equipment, review of ARS-funded foreign travel, and review of ARS funds provided through the SCA.
1. Identifying genes to improve breastfeeding rates for premature infants. It is well established that breastfeeding an infant has significant health benefits for both the infant and the mother and are related to the duration of breastfeeding. However women who deliver premature infants, and teenage or obese mothers have a high rate of lactation failure, and it is important to understand the molecular events that potentially lead to these failures. Researchers at the Children's Nutrition Research Center completed the analysis of the induction of gene expression from delivery through the first 6 weeks post partum in normal women. We were able to determine that a number of the genes normally expressed over the first 96 hour after birth in normal healthy women are not normally expressed in women with premature infants, or obese and/or teenage mothers, and we now have a genetic target that would lead us to attempt to determine the molecular and cellular mechanisms that might be suppressing or factors that might turn on these genes. These results may lead to an increase in the success of breastfeeding in all women, thus increasing the health and nutrition of their infants and decreasing the health risks of women who might otherwise have stopped breastfeeding their infants.
2. Mapping genomic regions associated with variation in lactation performance in the mouse. Genetic variation underlying milk production for lactation is not well defined. Children's Nutrition Research Center scientists identified 25 regions within the mouse genome that influence various levels of lactation performance. The regions contained 44 genes that could play an important role in determining milk production. Such findings are important as researchers discover the genetic background for what influences milk production levels.